JPH0745409B2 - Composition for releasing extension of biologically active polypeptide - Google Patents

Abstract

Non-aq. compsn. comprises at least 10wt.% of a biologically active polypeptide (I) and, as a continuous phase of the compsn., a biocompatible oil (II). Also claimed is a non-aq. compsn. of 15-40wt.% of a bovine somatotropin (Ia) chemically associated with zinc, 2-5wt.% of an aluminium mono- or di-stearate (IIIa) and, as a continuous phase of the compsn., a biocompatible vegetable oil. (Ia) has a particulate median vol. dia. of no greater than 5 microns and is present in a wt. ratio to the stearate of at least about 3. (Ia) chemically associated with non-toxic polyvalent metal is also new, pref. 0.3-2wt.% zinc.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to long-term released biologically active polypeptide compositions that can be administered parenterally to animals, methods of using these compositions, and certain of these compositions. Useful Metals-Bound Soft Tropins, and These Metals-
The present invention relates to a method for producing bound somatotropin.

BACKGROUND OF THE INVENTION Prolongation of the activity of some biologically active (bioactive) polypeptides can be achieved by administering a very small dose parenterally, while others have sufficient serum levels. Concentrations and / or short serum half-lives may result in substantial doses (eg, at least about at least about 1 week) to provide the desired biological activity for extended periods of 1 week or longer.
100 mg) should be administered. Somatotropin (growth hormone) is an example of such a polypeptide.

In order to prevent undesired rapid release in the bloodstream of animals, certain polypeptides are optionally hydrated retarders (anti-hydrating agents) or metals or metals that further reduce their solubility in body fluids. It was administered parenterally in a liquid vehicle containing the compound. To avoid the need for unacceptably large amounts of such vehicles, a substantial concentration of polypeptide in the vehicle is advantageous. However, most bioactive polypeptides are very viscous at substantial concentrations, and are therefore difficult to administer by injection or other methods at such concentrations. In addition, many commonly used anti-hydration agents increase viscosity, reducing the advantageous injectability of such compositions. For these and other reasons, (1) liberation fast enough to provide the desired biological effect in the animal, (2) liberation sufficiently slow to prolong the effect, The correct combination of (3) a suitable dose to release in the desired proportion over a long period of time, and (4) a sufficiently small volume and a sufficiently low viscosity for advantageous administration has usually been very difficult to achieve. .

Since each polypeptide, for example, differs in its three-dimensional structure and interaction with other substances, the possibility of achieving a long-term effective advantage with a high content of polypeptide in a suitable vehicle is theoretically predicted. It is impossible to show or substantiate.
However, in many cases, such long-term free compositions must be developed if the biological activity of the polypeptide is to be provided in a useful and economical manner.

DISCUSSION OF THE PRIOR ART Methods of achieving long-term release of bioactive agents have long been sought to reduce the frequency of treatment and / or minimize trauma to treated animals. Long-term release has been achieved in various ways for many such substances. One system is the use of oil solutions, which can be injected intramuscularly, subcutaneously or otherwise. In some cases low oil solubility materials were administered in oil suspensions.

For example, U.S. Pat. No. 2,491,537 discloses that Welhi gels with proteins such as pectin, cellulose compounds or gelatin and is released for up to 24 hours for penicillin suspended in oils (eg vegetable oils). . He suggests prolonged release of insulin and steroid hormones. In US Pat. No. 2,507,193 buckwalter gels with 5% aluminum monostearate (AlMS) and liberates up to 11 days using 300,000 units / ml procaine penicillin suspended in peanut oil in rabbits. I will disclose that. In U.S. Pat. No. 3,016,330 Jacobson discloses an AlMS-coated penicillin suspended in sesame oil.
Journal of Parenteral Science and Technology
Technology), 35 (3), 109 (1981), with 2% Al for chien
Discussing prolongation of biological activity of penicillin G procaine gelled by MS and suspended in vegetable oil, more than 2%
AlMS appears to have only a limited advantage for prolonging effective penicillin levels, and states that suspensions containing more than 2% AlMS are too viscous for practical use.

Oil suspensions have low molecular weights other than penicillin (M
W) Used for therapeutic substances. For example, Rutskmann et al. In US Pat. No. 3,676,557 up to 50% of pamoate of normorphinone gelled by AlMS.
Disclosed is a long-term active formulation of salt oil suspension. Siegel et al., In U.S. Pat. No. 4,016,273, discloses a long-term formulation of an oil suspension of up to 40% pamoate salt of oxazepine gelled with aluminum stearate.

A system for the long-term release of certain bioactive polypeptides has also been disclosed. For example, Anne Ciel has been described in US Pat.
No. 448, relaxin gelled by AlMS (about 2
%) Vegetable oil suspension. Ansiel showed that such suspensions provide comparable relaxation in oil without gelling agents (eg, 5-7 days), and heat treatment of suspensions containing AlMS produces longer-term effects ( (Until the 23rd) will be disclosed.

Geller in U.S. Pat. No. 3,869,549 discloses an injectable long-term release composition containing an "extremely small dose", eg, "mg fraction," peptide. Growth forms include, but a particular example is for water-soluble corticotrophins active for 7-8 days. In particular, Geller gelled with aluminum distearate (AlDS), suspended in peanut oil, or adsorbed on AlDS, and then dispersed in such oil, to form a composition containing an acid addition salt of the ACTH homologue. Disclose. In each case the homologue was only 0.03-0.
At a concentration of 1%, the peptide to aluminum salt is included in the injectable formulation of Geller in a weight ratio not greater than 0.5.

Compositions for the extended release of LH-RH hormone homologues are disclosed by Nester et al. In US Pat. No. 4,256,737. These compositions include hormone salts in vegetable oil, including polyvalent metal (eg, zinc) salts gelled with aluminum salts of fatty acids. The LH-RH homologue is administered at a concentration of 0.01-1% in the injectable composition.

Others have disclosed aqueous suspensions of metal salts or complexes of polypeptides for prolonging parenteral release. For example, Donini in U.S. Pat. No. 3,852,422 discloses water-soluble gonadotropins and long-term active aqueous suspensions of aluminum hydroxide or zinc hydroxide precipitation products. Since zinc is naturally present in pancreatic insulin, prolongation of insulin release in aqueous suspensions by interaction between insulin and various metals such as zinc, nickel, cobalt and cadmium has been investigated. U.S. Pat.Nos. 2,143,590, 2,174,862, and 2,882,
See Nos. 203, 2,920,014 and 3,102,077.

OBJECTS OF THE INVENTION It is an object of the invention to provide a composition comprising components which are useful in the extended release of biologically active polypeptides in an animal and which are biologically compatible with the animal.

Another object is to provide such a composition to the animal that has a release rate fast enough to produce the desired biological effect.

Another object is to provide such a composition with a release that is slow enough to maintain the desired biological effect for an advantageous extension period.

Another object is to provide such a composition containing a high enough content (appropriate dose) of the polypeptide to maintain the desired rate of release over such an extended period of time.

Another object is to provide such a composition with a sufficiently low volume to favor parenteral administration. This is especially important when the dose of polypeptide is unavoidably high.

Another object is to provide such a composition with the components and proportions selected such that the composition has a sufficiently low viscosity for advantageous injection or other administration. This is very important when injection of relatively large doses of polypeptide is required.

In some embodiments, such ingredients and proportions are intended to provide a sufficiently high viscosity to allow the animal to form a reservoir that favors longer term release. In many cases, this is difficult to achieve at the same time as the low viscosity objective above.

Other objects of the invention include providing methods of using such compositions, certain forms of somatropin that are advantageously included in such compositions, and methods of making such softtropins. These and other objects of the invention will be more readily apparent from the detailed description below.

SUMMARY OF THE INVENTION The above objective is to provide a substantially non-aqueous composition comprising a relatively high proportion of a bioactive polypeptide dispersed in a biologically compatible oil in an amount sufficient to form a continuous phase of the composition. I knew it could be achieved. In many embodiments, such objectives can be more advantageously achieved by using the polypeptides in combination with non-toxic metals. Optionally, such compositions may include anti-hydration agents to further prolong the release of the polypeptide. A relatively high ratio of polypeptide to anti-hydration agent is generally advantageous, especially for polypeptides which must be administered in relatively large doses and / or which substantially increase the viscosity of the composition. I got caught. Also, a composition having sufficient viscosity to help form a long-lasting reservoir in which a high loading of the polypeptide (optionally including an anti-hydrating agent) and the post-injection composition releases the polypeptide for a long time at an effective rate. I was also able to serve you.

DETAILED DESCRIPTION OF THE INVENTION Unless stated otherwise throughout the specification,% of the composition is by weight and the temperature is in degrees Celsius.

As used herein and in the claims, "substantially non-aqueous" is essentially anhydrous, or a low proportion of water that promotes release of the polypeptide in an animal to an acceptable degree. Means to include. This percentage of water will vary with each composition of the invention, but may be less than about 2% (and typically less than about 1%) in a manner that has such an effect on polypeptide release. It is normal.

"Non-toxic" herein refers to a composition of the invention that is reasonably safe and / or harmless when used in an amount and under suitable conditions for parenteral administration of such composition. Means an ingredient.

By "biologically active" or "biologically active" polypeptide is meant herein a polypeptide which, after proper parenteral administration to an animal, has a demonstrable effect on the biological process of the animal. The effects are hormonal, nutritional, therapeutic, prophylactic or other, simulated, supplemental,
Alternatively, it can block biological processes that occur naturally. Although there are a great variety of such effects and processes, food-stimulating growth stimulation, lactation, egg or pup production and / or feed efficiency can be mentioned as examples. Another example involves the production of wool, fur or other non-edible animal products. Although lower molecular weight polypeptides (eg, at least about 300) can be used, these polypeptides generally have a substantial molecular weight, eg, at least about
1000, typically at least about 4000, in most preferred embodiments at least about 9000, and in many more preferred embodiments at least about 18,000 to about 30,000 or higher molecular weight. Although the polypeptide is in active form in the compositions of the invention prior to administration to an animal, the term also includes herein polypeptides that exhibit biological activity after administration.

In many aspects of the invention, the polypeptides are administered in chemically unbound form. In many other embodiments, it may be advantageous to use the polypeptide in a form that has a lower solubility in the aqueous (eg, animal) fluid than the unbound polypeptide (eg, chemically bound to other substances). Is. For example, a polypeptide is predominantly (eg, completely) chemically bound to a non-toxic metal or is an ester, amide or other form that provides a desired biological activity and does not have unacceptable side effects. . When chemically bound to such metals,
The metal may be present as the metal itself (eg, in a metal salt of the polypeptide or in complex with the polypeptide) or in the form of a salt or complex of the metal with one or more other anions.

Monovalent metals (eg sodium or potassium) may be used to advantage in some aspects of the invention, while polyvalent metals are often preferred to be used. Examples of such polyvalent metals include zinc, iron, calcium, bismuth, barium, magnesium, manganese, aluminum, copper, cobalt, nickel, cadmium and the like.
In some highly preferred embodiments, such metal-binding polypeptides are the reaction products of these metals, eg, in ionic form, with the soluble polypeptide. The metal to polypeptide ratio will vary with the number of active sites on the polypeptide that bind such metal during the formation process. For example, the metal binds to some or all negatively charged amino acid (eg, aspartic acid or glutamic acid) residues or carboxy terminal groups of the polypeptide. Some or all of the metal binds to the polypeptide salt, is occluded within the folded, crystalline or amorphous form of the polypeptide, or binds as a cationic bridge between at least two polypeptide molecules.

If the metal is multivalent, its valence is, in some cases,
For example, it only partially chemically binds to the polypeptide due to steric hindrance. In such cases, the remaining valences of the metal chemically bond with other anions. In many desirable embodiments, the metal does not chemically bind salts with low water solubility with other anions that form with the metal in a substantial proportion. When the metal partially chemically bonds with other anions,
These other anions (organic or inorganic) are water-soluble salt-forming anions such as Br ⁻ , Cl ⁻ , I ⁻ , ▲ SO ⁼ ₄ ▼
Or, it is often desirable to be selected from CH ₃ COO ⁻ when the metal is zinc. Monovalent anions such as Cl ^- are generally preferred.

The novel and preferred metal-binding polypeptides of the present invention include zinc bound somatotropin. In some cases
These contain up to about 5% or more zinc by weight standard for somatotropin. However, it is desirable to include less than about 2%, and in some cases less than about 1% zinc (same criteria) to minimize the chance of undesired injection site response in the animal. In many preferred embodiments, these polypeptides will contain at least about 0.3% (usually at least about 0.5%) zinc (same criteria), although lower% zinc is appropriate in some cases.

Examples of other polypeptide salts useful in the present invention are (a) with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid or nitric acid, or with organic acids such as acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid. Acids formed by acids, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, naphthalene-disulfonic acid or polygalacturonic acid. Addition salts, (b) salts with polyvalent organic cations such as N'-diverdylethylenediamine or ethylenediamine, and (c) combinations of two or more salts of the above type, such as tannic acid, zinc.

Preferred are zinc, iron, calcium, magnesium,
Salts of manganese, sodium, potassium and mixtures thereof. Particularly preferred salts are zinc, sodium or potassium salts, especially when the polypeptide is somatotropin. Very advantageously, these salts interact with the oil when administered to the biocompatible oil in the proportions used in the compositions of the invention and, surprisingly, at effective levels of polypeptide release. It has been found to form a matrix or other structure that enhances elongation. This unexpected finding is noted both by subcutaneous and intramuscular administration and appears to make a significant contribution to the efficacy of many of the compositions of the invention.

The compositions of the invention are also useful for extending the release of conjugated or unconjugated polypeptides. These conjugated polypeptides are lipoproteins, such as beta lipoproteins; glycoproteins, such as gamma globulin; phosphatans, such as casein; homotans, such as hemoglobin; flavonoids,
Examples include succinate dehydrogenase; and metalloproteins such as ferritin and alcohol dehydrogenase. Mixtures of polypeptides of these or any other of the above forms are considered to be within the invention described and claimed herein.

In many attractive embodiments, the polypeptides administered to the compositions of the invention are poorly water soluble (ie, room temperature water required for dissolution of 1 part polypeptide is at least 100 parts). In many desirable embodiments, they are poorly soluble in the biocompatible oils used herein. Ie about 2 at room temperature
It does not dissolve at concentrations higher than%, and it is desirable to keep it about 1
Not higher than%. In some cases, for example, in compositions containing various somatotropins, the polypeptide is slightly water soluble and substantially oil insoluble.

As noted above, the composition of the present invention comprises a biocompatible oil as its continuous phase, i.e., an oil that does not have an unacceptable adverse effect on the polypeptide, animal, or animal In some cases, the animals consuming each of these products are included. Desirably, such oils are of low acidity and are substantially not rancid. As used herein, "oil" means a fatty oil or fat that is liquid at the body temperature of the animal. Such oils are not more than about 40 °, preferably about 35 °
Melts below, or at least begins to melt. Oils that are liquid at about 25 ° make injection or other administration of some compositions of the present invention advantageous. In some cases, polyunsaturated (eg, partially hydrogenated) oils are advantageous for greater biocompatibility for animal or other reasons.

In a preferred embodiment, biocompatible oils essentially triglycerides, namely the long chain glycerol (typically C ₈ -C _24,
Preferably of C ₁₂ -C ₁₈₎ a mixture of fatty acid esters or triglycerides and their fatty acid (preferably very small portion, e.g., about 10% less than the fatty acid). In some embodiments, other trihydroxy or polyhydroxy compounds can replace glycerol. Particularly preferred oils include vegetable oils such as olive oil, sesame oil, peanut oil, sunflower seed oil, soybean oil, cottonseed oil, corn oil, safflower oil, palm oil, rapeseed oil and mixtures of these oils. Sesame oil and peanut oil are highly preferred for many embodiments. Oils of animal or mineral origin, or synthetic oils (including long chain fatty acid esters of glycerol or propylene glycol) can also be used provided they are sufficiently biocompatible.

In most embodiments, these oils make up the predominant part by weight of these compositions. The continuous phase of biocompatible oils in most cases desirably has relatively uniformly dispersed finely divided, discrete particles of the polypeptide (eg, in suspension). The upper limit of the polypeptide content is when the oil can no longer be present as the continuous phase, since substantially all of the polypeptide of the composition cannot be completely wrapped due to insufficient oil.

It has been found that the use of high levels of polypeptide in these compositions gives surprising and unexpected results, even when the viscosity thereby increases substantially. Furthermore, it has been found that at such high contents, there is often an interaction between the polypeptide and the oil that favors prolonged release of the polypeptide from long-term persistent storage. This interaction is often enhanced when the polypeptide binds a metal, as described above.

Compositions of the invention desirably include the polypeptide at high levels, eg, at least about 10%. Even higher contents of polypeptide, eg, at least about 15%, are often desirable, especially with softtropin and other polypeptides having substantially the same properties. A content of about 20% or higher, such as up to at least about 30% or about 42% or higher, is found in parenteral injectable compositions containing somatotropin (eg, bovine), particularly somatotropin with polyvalent metals such as zinc. It can be used advantageously when combined.
Such compositions can provide extended release of somatotropin (as measured by blood flow in cattle or other animals) for up to 30 days or longer.

To the best of our knowledge, no substantially non-aqueous composition comprising a polypeptide content of about 10% has been suggested in the prior art. In these prior art, oil formulations are limited to very low polypeptide content, ie, no more than about 2% (US Pat. Nos. 2,964,448, 3,869,549 and 4,25).
See 6,737 specification).

In addition to biocompatible oils, the compositions of the present invention can include "anti-hydrating agents". As used herein, "anti-hydrating agent" means delaying the hydration of a given composition of the invention, or a polypeptide and / or biocompatible oil therein, whereby it is present after administration to an animal. It means to reduce and / or stabilize the rate of release of the polypeptide from the composition. Various non-toxic anti-hydrating agents are known. An example is a "gelling" agent. This gives the body of the oil greater viscoelasticity (and thus greater structural stability) when dispersed and in some cases when heated and dissolved in the oil, thereby making the oil more susceptible to aqueous (eg body) fluids. Slow the penetration of.

The exact mechanism of action of these agents in the present invention is not safely understood. Some known "gelling agents are those in which the oils containing these agents are not heated to enhance their gelling effect, or even once the gel once formed is substantially removed (eg shearing forces). It has been found to provide the desired anti-hydration effect even when applied, and various anti-hydration agents that do not have the substantial ability to gel oils are suitable for use in the present invention.
Magnesium stearate is an example.

Examples of anti-hydrating agents are various salts of organic acids, such as fatty acids having from about 8 (preferably at least about 10) to about 22 (preferably up to about 20) carbon atoms, such as lauric acid,
It includes aluminum, zinc, magnesium or calcium salts such as palmitic acid and stearic acid. These salts are mono-, di-, or tri-substituted depending on the valence of the metal and the degree of oxidation of the metal by the acid. Aluminum salts of such fatty acids are especially useful. Aluminum monostearate and aluminum distearate are particularly preferred anti-hydrating agents. Others useful are aluminum tristearate, calcium monostearate,
Includes calcium distearate, magnesium monostearate, magnesium distearate and corresponding palmitates, laurate and the like. Oil + in many embodiments
Concentrations of these anti-hydrating agents under this drug standard are advantageously from about 1 to about
It is 10% (generally about 2 to about 5%). However, other concentrations are suitable.

Generally, both the polypeptide and the anti-hydration agent tend to increase the viscosity of the composition of the present invention. For many polypeptides, especially relatively high molecular weight and / or conjugates of secondary or tertiary structure, this problem can be overcome by using a relatively high polypeptide to anti-hydration agent weight ratio. In the present invention, this ratio is generally at least about 1, more typically at least about 3, more typically at least about 4, and most usually at least about 6. Although generally less critical due to the viscosity of the composition, this ratio is generally no greater than about 40, and more typically about 20.
Not greater.

It has been found that using such ratios, beneficially long effective polypeptide release is obtained, even for high viscosity compositions inherent in relatively high polypeptide concentrations. Even more surprising is the fact that in some such compositions, the free fraction actually increases when the polypeptide content (and thus the composition viscosity) increases.

The composition of the present invention is used for prolonging the release of a polypeptide in animals including humans and other primates, particularly mammals. Polypeptides useful in treating these animals are, for example, chickens,
Bird hormones that treat turkeys, humans, cows, pigs,
Includes mammalian hormones for treating sheep and goats, and aquatic animal hormones for treating fish. Particularly useful polypeptides include growth promoting hormones and lactation enhancing hormones. These hormones include somatotropin, which is useful in increasing red meat-to-fat ratio, feed efficiency and milk production in various mammalian species including cows (eg dairy cows), sheep, goats and pigs.

As used herein, "somatotropin" means a polypeptide having substantially the same biological activity and chemical structure as somatotropin produced in the pituitary of an animal.
Such somatotropin natural somatotropins produced by somatotropin production cell pituitary, and another containing the somatotropin represented by Ikori (E.Coli), genetically transformed microorganisms such as other bacteria or yeast. Such otherwise produced somatotropin has the same amino acid sequence as native somatotropin, or has one or more changes in the amino acid sequence that provide increased biological activity or some other advantage. It is a homologue. Polypeptides with which the present invention is particularly useful are bovine and porcine softtropins, such as microbially represented bovine and porcine somatotropins and prolactin from bovine, porcine or other animals, growth hormone releasing factor, insulin uniform growth factor and the like. . These polypeptides optionally have N-terminal methionine residues, such as methionine produced by microbial translation of the ATG start signal in the gene for the polypeptide. However, in some cases, such methionine residues of the polypeptide may be reduced by about 20% to reduce the tendency of the animal to degrade the polypeptide to prevent foreign body protection.
It is desirable to have less (preferably no more than about 10%) formyl-methionine.

Injection observations of the compositions of the invention show that substantial amounts of the polypeptide are initially released upon injection. This means "rupture" which is believed to be the result of the increased surface area caused by injection or other administration. In some cases, for example, a modest rupture is desirable to activate the desired biological effect. A property useful in formulating the compositions of the present invention is that the initial rupture level, as determined by measuring the polypeptide concentration in the serum of treated animals shortly after administration, and the subsequent determination of the polypeptide concentration in animal serum. And the extended release level as measured by For purposes of this invention, rupture level is the serum polypeptide concentration 24 hours post injection and extended release level is the serum polypeptide concentration 14 days post injection. These concentrations are used to calculate a "burst-extended free ratio" which is generally considered to be about 1.2 to about 6, for example about 1.5 to about 3.

Another property useful in evaluating and formulating compositions of the present invention is "injection capacity" which is a measure of how well the composition flows through a hypodermic needle. If the polypeptide particles are too large or the composition is too viscous, excessive pressure is required to force the composition through such a needle. For purposes of this invention, "injection capacity" is 173 psi (1193KP
When the pressure in a) is applied to the composition of a syringe fitted with a needle, measure the time for the volume of the composition of the invention to pass through an 18 gauge hypodermic needle having an ID of 0.033 inches (0.838 mm) and a length of 4 cm. It is measured by The "injection capacity" for the composition of the present invention is preferably at least 0.03 ml / sec (ml / sec). Preferably such injection capacity is higher, eg at least about 0.1 ml / sec, even more desirably at least about 0.3 ml / sec.

The composition of the present invention is produced by adding the polypeptide to the oil alone or the oil having the anti-hydration agent suspended or dissolved in the oil. Often it is advantageous to dissolve the anti-hydrating agent and provide a gelled oil. When the composition of the present invention is produced by a method starting from forming a gelled oil, a gelling agent such as a fatty acid aluminum salt may be added as a powder to the stirring oil quantity to obtain a suspension of this powder. The stirring suspension is preferably heated to at least the melting point of the fatty acid salt (eg AIMS is at least 155 °) at which the fatty acid salt dissolves in the oil. Lower temperatures can be used than if the gelling agent or other anti-hydration agent is adequately dissolved. Sufficient and continuous agitation avoids agglomeration of fatty acid salts and keeps the dispersion. Generally heating and stirring should be continued until the suspended salt is completely dissolved. Keep stirring for additional hours,
It is often desirable to ensure complete dissolution.

The oil solution of the fatty acid salt is then cooled, for example to room temperature, where a fairly stable gel structure forms. The gel should be stored by vacuum or desiccant to avoid contamination by water, which adversely affects the gel structure.

Polypeptides then have unacceptable opposite effects (eg,
Add to oil at any temperature (eg room temperature) to avoid denaturation. For example, bovine soft tropin was added to such oils at temperatures of about 4-125 ° without adversely affecting biological activity. This addition of the polypeptide is preferably done under vacuum to avoid contamination by water or air bubbles. Such addition is preferably accomplished by slowly adding the finely divided polypeptide to the oil with high shear mixing to provide a uniform dispersion of the polypeptide particles. It is often desirable to reduce the size of polypeptide particles, such as polypeptide suspensions
It can be achieved by using a pole mill mixed with a large amount of stainless steel balls having a diameter of 0.3-0.6 cm. This is advantageously done simultaneously with such dispersion in the lower part of the vessel where high shear mixing takes place. In particular, highly loaded poly that is difficult to reduce in size to particles having a median particle size not greater than about 15 microns by volume criteria (ie, 50% of the volume of particles having a diameter not greater than about 15 microns). Peptides are advantageous. It has been found that the use of polypeptide particles of small size (eg, particles of medium size not greater than about 10, preferably about 5 microns) is desirable to increase the injectability of the compositions of the present invention. By operating such a ball mill, the polypeptide particles can be advantageously reduced to a preferred intermediate particle diameter of no greater than about 5 microns. The composition of the invention is then recovered from the ball mill by filtration and, preferably under vacuum.

As noted above, the compositions of the present invention are so useful as to be attractive by parenteral administration, eg, intraperitoneal injection, or more preferably subcutaneously or intramuscularly. The extended release period is the period in which the polypeptide is provided at the rate required for the desired biological effect, generally at the rate indicated by the polypeptide concentration in the circulating blood stream of the animal. Depending on the particular polypeptide and biological effect, a release extension period of at least about 7 days is desirable. In other cases, it may be at least about 15 days, or more desirably for many applications at least about 30 days, or at least about 60 days or longer. According to the invention, a composition comprising bovine somatotropin bound to zinc is provided in serum of lactating cows injected with a 2.5 ml dose for at least about 7 days, at least about 12 ng / ml.
It was found that the mean bovine somatotropin concentrations of
This is very advantageous for the purpose of milk production and / or enhancement of cattle feed-milk conversion efficiency. To provide an effective dose of bovine somatotropin for the treatment of dairy cows, for example to increase milk production, a composition comprising at least about 300 mg of zinc-bound somatotropin for at least about 15 days,
Desirable to increase serum levels of bovine active somatotropin. The zinc-bound somatotropin is preferably included in a sufficiently high concentration (eg, at least about 15%) to provide a small volume of composition (eg, less than about 10 ml, eg, about 1 to about 3 ml) for each administration. Being able to be used is a very attractive feature of the invention.

Of course, other substances can be added to the composition as long as these substances do not unacceptably block the desired extension of the polypeptide at effective levels. For example, to reduce or prevent foreign body reaction (eg, non-allergic) effects,
Alternatively, it may be desirable to add anti-inflammatory or other additives to the composition of the invention to counteract it. Such additives include steroids and / or non-steroidal anti-inflammatory agents, which are preferably at low levels to avoid some systemic effects, but at levels sufficient to reduce local inflammation. Included in the composition.

Production of Metal-Binding Biologically Active Polypeptides Polyvalent metal-bound polypeptides can be produced by reacting a lytic polypeptide with a non-toxic metal (eg, zinc) ion. Generally, the metal-binding polypeptide is a metal, optionally in the form of a poorly water-soluble salt thereof, but is generally preferably added as a water-soluble salt thereof (eg, zinc chloride) to a buffer solution of the polypeptide to bind the metal-bound polypeptide. Is prepared by precipitation.

Often solubilizing compounds, such as urea, guanidine, etc., are included in the solution to aid in the dissolution of the polypeptide, especially the slightly water-soluble polypeptide. Often, the concentration of solubilizing compound and / or pH of the solution are critical to maintaining the bioactive structure of the polypeptide.

Moreover, the pH of the solution is generally critical for recovering the metal binding polypeptide, which generally forms as a precipitate.
For example, there is a critical range of pH due to the isoelectricity of the polypeptide. It is desirable to keep the temperature low to avoid denaturation, eg, generally not above about room temperature.

Depending on the purity of the polypeptide utilized, pyrodiene removal and / or sterilization may be desirable. Pyrodiene (eg, endotoxin), if included, can be removed by contacting the polypeptide solution with an ion exchange resin. Many pyrodiene binds to positively charged sites, such as cation exchange resins. Some pyrodiene binds to negatively charged sites. Therefore, the mixed layer of ion exchange resin beads is useful for ensuring sufficient removal of pyrodiene. The polypeptide solution can be sterilized and non-sterile foreign material such as bacteria or loose contaminants from previous treatments can be removed by filtration through a fine filter, eg 0.2 micron mesh.

The pyrodiene-removed, sterile polypeptide solution is then contacted with a non-toxic metal solution to precipitate the metal-binding polypeptide, typically preferably forming a suspension. The metal loading rate affects the particle size of the metal-binding polypeptide produced. Metal addition can be controlled by adjusting the metal concentration, volumetric flow rate and / or dispersion rate of the additive solution.

Typically, metal-binding polypeptide suspensions can be diluted to reduce the tendency for particle size to increase, eg, by aggregation. The metal-binding polypeptide is then subjected to multiple centrifugations and washings to remove excess metal and anions, and then recovered, followed by lyophilization.

Polypeptides bound to monovalent metals (eg sodium or potassium) can be prepared by lyophilization of the polypeptide and metal ion solutions.

Preparation of Metal-Binding Biologically Active Somatotropin In a more specific example procedure described herein, somatotropin (eg, bovine) is dissolved in a variety of buffer solutions. It is preferably dissolved in an aqueous urea solution buffered with tris (hydroxymethyl) aminomethane (TRIS) or other suitable buffer. The desired upper limit for urea concentration is typically 6M, and in some cases a urea concentration of about 4.5M is preferred.
Even lower urea concentrations, such as 3M or 2M or 1M, can be used except that the solubility of somatotropin is low.

The pH of the buffered urea solution is preferably about 7 to about 10.5. these
Recovery of somatotropin precipitated from solution between pH limits is generally at least about 60%. In general, higher recovery (eg, at least 90%) can be achieved with a pH of about 9 to about 9.5. The temperature of the solution throughout the precipitation should be low enough to prevent oligomerization of softtropin. Generally, such temperatures are desirably about
Less than 10 °, more preferably less than about 5 °.

Monovalent metal - binding polypeptides in order to provide the solution (to Pairojien removal and sterilization of the above) diafiltration (or dialysis) processing, urea NaHCO ₃ solution of the metal bicarbonate (e.g. 25 mM, pH 9. 5) Or replace with other suitable salt. Multiple exchanges with bicarbonate solution are preferably performed to ensure complete exchange of urea. The solution is then further diafiltered with water to remove excess NaHCO ₃ .
This is indicated by the onset of MBS precipitation. Sodium-somatotropin is recovered and lyophilized to obtain powder of somatotropin sodium salt.

To provide the polyvalent metal-bound somatotropin, the solution (pyrodiene-removed and sterilized above) is contacted with a polyvalent (eg, zinc) salt. It has been found that the use of a 1M solution of zinc chloride produces an acceptable precipitation of zinc-somatotropin from a 4.5M urea solution of somatotropin. However, higher or lower concentrations of chloride can be used. It is preferable that the ZnCl ₂ solution is added gently while stirring the somatotropin solution, for example, by titration.

With continued addition of the ZnCl ₂ solution, the somatotropin solution becomes grayish white first and then pearl white when the stoichiometric amount of ZnCl ₂ is added. For example, 4 ml of 1M Zn in 400 ml of pH 9.5 solution containing about 20 mg / ml somatotropin.
By adding 0.09MTRIS of Cl ₂ and 4.5M urea, pearls white zinc - somatotropin suspension pen Ji is formed. About 10ml of additional ZnCl ₂ (e.g. 1MZnCl ₂ solution
Up to) can be added to ensure complete precipitation.

Secondly, it is often desirable to dilute the suspension to reduce the tendency of the precipitate to increase in size. It has been found that diluting to about 1 M urea with about 3.5 volumes of water is sufficient to prevent agglomeration of zinc-somatotropin particles. Urea, TRIS
And recovered by diafiltration (or multiple centrifugation and washing) to remove zinc and chloride ions, followed by lyophilization to give a powder, generally with a particle size of less than 10-20 microns.

If somatotropin is bovine, the particles are generally about
0.3 to about 1% zinc (about 1 per molecule of somatotropin
~ 4 molecules of zinc). If the proportion of zinc added increases,
Higher amounts, eg up to 4-5%, are found during precipitation. Such high amounts are due to the addition of zinc to the active site of somatotropin, such as additional aspartic acid and / or glutamic acid residues or possibly histidine residues, or the carboxy terminal group of the polypeptide. This theory of zinc binding is not intended to limit the scope of the invention. Precipitation using essentially the least amount of zinc is generally considered preferable.

The following disclosure is provided to illustrate particular embodiments and aspects of the present invention, but is not meant to limit the scope of the invention.

Example 1 This example illustrates the preparation of zinc-bound bovine somatotropin of the present invention.

Bovine somatotropin (MB with methionine N-terminal group
S) is the description by Siebrug et al., Here incorporated by reference, Efficient Bacterial Expressions of Bobbin and Pocine Grouse.
Hormone (Efficient Bacterial Expression of Bovine
and Porcine Growth Hormone) ", 2 (1) DNA 37-45
(1983). Somatotropin was recovered in useful form by lysing the bacterial cells and then separating the somatotropin from the bacterial cell debris.

In one method of recovering MBS, bacteria were killed by treating the fermentation broth with enough 50% sulfuric acid to reduce the pH to 1.7. Broth was neutralized with NaOH and centrifuged, leaving behind a bacterial paste, which was suspended in urea, homogenized, cooled to about 4 ° C (this temperature was maintained until MBS lyophilization below) and centrifuged. Wash three times, dissolve in guanidine hydrochloride (7M), centrifuge to remove insolubles,
Filtered, passed through a G25 Sephadex column that allowed guanidine to react with urea, filtered, and then passed through a DE52 ion exchange column. The effluent volume was reduced by about 30 × by hollow fiber ultrafiltration. The concentrated solution was passed through a G75 Sephadex chromatography column and through another hollow fiber volume reduction step, then firstly urea to NaHCO ₃ solution and then to distilled water to precipitate MBS. It was dialyzed against. The precipitate was lyophilized, NH _2-met shown in the publication by Seeberg et al
-Phe (1) -pro (2) ... leu (126) ... phe (190) -CO
A white solid containing a polypeptide (MBS) having an OH amino acid sequence was obtained.

This MBS is dissolved in 4.5M urea and is 0.09 MTRIS at 4 ° and pH 9.5.
The solution was dissolved at 21.5 mg MBS / ml. For 1 ml of sterile MBS solution
MBS by mixing 0.2 g of anion / cation mixed ion exchange resin beads (Bio-Rad AG-501 x 8)
The solution was pyrodiene removed. The mixture is about 10 minutes 4
Stirred at 0 ° and then filtered through a 1 micron nylon filter to remove beads containing adsorbed pyrodiene.

Pyrodiene-removed MBS were sterilized by passing the solution through a radiation-sterilized, pleated capsule with a 0.2 micron mesh to remove non-sterile foreign material such as bacteria or free contaminants from previous treatments.

MBS was converted to zinc salt (ZnMBS) by adding 1M ZnCl ₂ while stirring the MBS solution with pyrodiene removed. ZnMB
The S precipitate contained about 1% zinc. The solution containing ZnMBS solids was then diluted to 1M urea concentration with sterile pyrodiene removal water.

ZnMBS was recovered by centrifugation at 10,000 × g for 30 minutes while keeping the solution at 4 °. ZnMBS is 50m using high shear mixing
The cells were suspended in sterile pyrogen-free water at gZnMBS / ml. ZnMBS
Is harvested again by centrifugation at 10,000 xg for 30 minutes, resuspended in sterile pyrodiene-removed water at 50 mg ZnMBS / ml using high shear, then freeze-dried to sterile ZnMBS as a white feathery powder.
Got

Example 1A This example illustrates another method of making ZnMBS.

Pyrodiene-removed, sterile 21 mg MBS / ml solution in 4.5 M urea, 0.05 M TRlS at 10 °, pH 8.8, was recirculated through a holding tank with a positive displacement pump. 1M ZnCl ₂ was pumped in until the concentration of the solution was 0.01 M ZnCl ₂ which produced a precipitate of ZnMBS. Diluted water further produces ZnMBS precipitates 1
Added to provide a concentration of 0 mg MBS / ml. Next, the suspension of ZnMBS is suspended until the concentration reaches 40mgMBS / ml.
It was circulated at 25 ° through a diafiltration hollow fiber membrane with pores that pass molecules up to 100,000 MW. In that case, water was added to balance the membrane filtrate rate until essentially all of the zinc, urea and TRlS had been removed from the suspension. Water addition was stopped to concentrate to about 80 mg MBS / ml. The concentrated suspension was then lyophilized to give a dry, white ZnMBS powder with a particle size in the range of 0.5-11 microns.

Example 1B This example illustrates the preparation of Na-bound bovine somatotropin.

Pyrodiene-removed, sterilized 21.5 mg / ml solution in 4.5 M urea, 0.05 M TRlS at 4 °, pH 9.5 was dialyzed to remove urea first.
The aHCO ₃ solution was exchanged and then distilled water. When MBS began to precipitate, the water exchange was stopped. The solution is then filtered through a 0.2 micron filter to remove MBS precipitate and lyophilized to remove sodium salts (Na
MBS).

Example 2 This example illustrates the preparation of a composition of the present invention containing Zn-linked somatotropin.

Sesame oil (Fisher NF grade) was added to a 3-neck round bottom flask. Anti-hydration agent (AlMS) was added to AlMS
%. The flask was placed in a 155 ° oil bath and stirred to disperse the AlMS as soon as possible. Continue stirring for 20 minutes,
Meanwhile, AlMS was completely dissolved in oil. The flask was removed from the bath, kept under vacuum and cooled to 25 °. On cooling the solution converted to a thick gel. The cooled gel was fed to a ball mill with a high shear stirrer in a bed of stainless steel balls having diameters of 1/8, 3/16 and 1/4 inch (0.32, 0.48 and 0.64 cm). A vacuum is applied to the mill to produce a ZnMBS powder (produced as described in Example 1) with a composition of 40% ZnMBS (ZnMBS vs.
The mixture was added gently using a screen feeder until it contained the AlMS weight ratio of 13.3). Stirring continued for 6 hours, during which the volume median diameter of the ZnMBS particles was reduced from 20 microns to 5 microns. The resulting substantially non-aqueous ZnMBS gelled oil suspension was separated from the steel balls by filtration.

Example 3 This example illustrates the effective use of the composition of the invention to prolong the release of a polypeptide, bovine somatotropin, to enhance milk production in lactating dairy cows.

The substantially non-aqueous composition is essentially as in Example 2.
It was prepared by dissolving 5% AlMS in sesame oil heated to 155 ° C. The oil was cooled to form a gelled oil. Until the composition contains 32% ZnMBS in the oil continuous phase (ZnMBS vs. Al
MS weight ratio 9.4) ZnMBS was dispersed in oil and ground. A syringe with an 18 gauge, 1.5 inch (3.8 cm) long needle
2.54 g (2.5 ml) of composition was loaded to provide a dose containing 805 mg ZnMBS. The composition had an injection capacity of 0.36 ml / sec.
A blank composition of 5% AlMS in sesame oil without polypeptide was also prepared and loaded with 2.4 g in the same syringe.

The composition was injected into 23 Holstein dairy cows during the second or third three-month period of these second (or subsequent) lactations. Cattle were randomly organized into 4 groups of 5 or 6 cows. Two groups were injected intramuscularly in the buttocks (1M), one group was injected with a ZnMBS-containing composition, and the other group (control group) was injected with a blank composition. Similarly, the other two groups were injected subcutaneously (SQ) in the upper part of the scapula with a composition containing ZnMBS or a blank composition.

The cumulative least squares mean (adjusted by covariate for pretreated milk yield) for average milk production is shown in Table 1. Milk production in this table is expressed in kg of milk per day. As shown in Table 1, a single 1M or SQ injection of the advantageously administered composition of the present invention provides a rapid and prolonging improvement in milk production at a very high statistically significant level.

Blood samples were analyzed for bovine somatotropin normally contained in the circulatory system of cattle without administration according to the invention. A representative analysis by radioimmunoassay (RlA) is shown in Table 2, where bovine somatotropin concentration in plasma is expressed in ng / ml.

Example 4 The present invention uses various substances in the composition of the present invention to exemplify the efficacy of the composition of the present invention for extending the release of polypeptide (MBS) in animals.

In this example, the ZnMBS composition has the following components: Biocompatible oil: Sesame oil or peanut oil Anti-hydration agent: Oil + 3% of AlMS or 5% of AlMS Polypeptide content: 20%, 30% of total composition Or 40% Zn
Formulated essentially as disclosed in Example 3 using a combination of MBS. AlMS was dispersed in oil. After heating to 155 ° and holding at 155 ° for 15 minutes, the dispersion was cooled to 25 ° to form a gelled oil. ZnMBS was added and dispersed with a high shear mixer (Polytron homogenizer) to form a gelled oil suspension of ZnMBS. Suspension
It was loaded into a tuberculin syringe with an 18 gauge hypodermic needle.

The compositions of Table 3 were administered to 16 groups of 8 immunosuppressed spray gourdery (IFS-D) female rats by subcutaneous injection at the dorsal scapular site.

Blood samples were analyzed by RIA for bovine somatotropin. The analysis in Table 4 is ng / ml of plasma. These plasma levels are shown in Table 4 for blood samples taken before injection on day 0 (day of injection). Some baseline measurements for the rats of Examples 4-7 are higher than some baseline and free polypeptide measurements for the cow of Example 3. This was partly due to the interspecific differences in normal somatotropin levels and partly because the RIA of Example 3 was more accurate.

Example 5 This example illustrates the efficacy of the composition of the invention on the extended release of polypeptide (MBS) using other fatty acid aluminum salts as anti-hydrating agents. In these compositions, aluminum monolaurate (AlML) and aluminum monopalmitate (AlMP) were utilized as anti-hydrating agents with sesame oil and peanut oil.

In this example, a gelled oil containing 3% AlML or AlMP was prepared essentially as in Example 4. ZnMBS was suspended in gelled oil at a concentration of 30% of the total composition (weight ratio of ZnMBS to AlML or AlMP 14.3). Each composition was administered in the doses shown in Table 5 in a group of 8 IF
S-D rat was injected.

Analysis of blood samples taken from the rat on the indicated day post-injection was for bovine somatotropin concentrations shown in Table 6. In this table, the reading after day 0 is the baseline for the analysis.

Example 6 This example illustrates the efficacy of the composition of the present invention on free extension of polypeptide (MBS) utilizing olive oil or corn oil.

In this example, a gelled oil was prepared essentially as in Example 4 utilizing 3% AlMS with AlMS + oil as the reference. 30% or
40% ZnMBS suspension I at 8 doses as shown in Table 7
Two groups of FS-D rats were injected.

Analysis of blood samples taken from the rat on the indicated day post injection gave the bovine somatotropin concentrations shown in Table 8. The reading after day 0 in this table is the baseline for the analysis.

Example 7 This example shows that peanut oil contains about 10% of the polypeptide, namely MBS
2 illustrates a composition of the present invention including ZnMBS and ZnMBS. This example further illustrates that the prolonging effect of a polypeptide can be enhanced by using a metal-binding polypeptide and by using an anti-hydration agent. The injectable composition shown in Table 9 was prepared essentially as in Example 4.

Each composition was injected subcutaneously into one group of 8 IFS-D rats in the same volume of 300 microliters. Analysis of blood samples taken from the rat on the indicated day after injection showed the plasma concentrations shown in Table 10. In this table, the 0 day reading is the baseline for the analysis.

A comparison of the results for groups 30 and 31 illustrates the increased prolongation of MBS release for at least 7 days due to the use of bound polyvalent metals. A comparison of the results for groups 32 and 33 illustrates the increased prolongation of MBS release when MBS binds to these polyvalent metals due to the use of anti-hydration agents.

Example 8 This example contains the following oils: sesame oil, peanut oil, corn oil, olive oil, safflower oil, cottonseed oil, palm oil, rapeseed oil and soybean oil without anti-hydrating agents and 10% bovine somatotropin. 1 illustrates an inventive composition. Each different oil has the following temperature: 4
Hold at °, 25 °, 50 °, 75 °, 100 ° and 125 °.
ZnMBS are dispersed in each oil and milled as in Example 2 until the concentration reaches 10%. Attrition continues until somatotropin has an intermediate particle size not greater than 15 microns.
Each composition has an injection capacity of greater than 0.1 ml / sec.

Example 9 This example illustrates a composition of the invention prepared as in Example 8 except that AlMS was dispersed in each oil to a concentration of 5% on oil + AlMS standard prior to the addition of somatotropin. These compositions have an injection capacity of greater than 0.1 ml / sec.

Example 10 This example illustrates the composition of the present invention prepared as in Example 8 except that the addition of somatotropin was continued until the composition contained 40% bovine somatotropin. Dispersion and milling continue until somatotropin has a median particle size no greater than 15 microns. These compositions have an injection capacity of greater than 0.03 ml / sec.

Example 11 This example illustrates a composition of the present invention prepared as in Example 10, except that AlMS was dispersed in each oil to a concentration of 5% based on oil + AlMS prior to the addition of somatotropin. These compositions have an injection capacity of greater than 0.03 ml / sec.

Example 12 This example contains 10 of the following oils: sesame oil, corn oil, olive oil, safflower oil, cottonseed oil, palm oil, rapeseed oil and soybean oil.
1 illustrates a composition of the present invention comprising% bovine somatotropin.
Each oil is heated to 160 ° and stirred to help dissolve AlMS. Once the 1% AlMS has dissolved, each oil cools to 25 °. Zn
The MBS is dispersed in a cooling oil and ground as in Example 2 until the concentration reaches 10% and the median particle size is reduced to less than 15 microns. Each composition has an injection capacity of greater than 0.1 ml / sec.

Example 13 This example illustrates a composition of the invention made as in Example 12, except that the addition of somatotropin was continued until each composition contained 40% somatotropin. The composition is ground as in Example 2 until somatotropin has a median particle size not greater than 15 microns. 0.03 for each composition
It has an injection capacity of more than ml / sec.

Example 14 This example oil + AlMS has the following oils with AlMS dissolved in oil to 5% concentration: Sesame oil, peanut oil, corn oil, safflower oil, cottonseed oil, palm oil, rapeseed oil and soybean oil 10% beef 1 illustrates a composition of the present invention containing somatotropin. Each oil is heated to 160 ° and stirred to help dissolve AlMS. When the AlMS dissolves, each oil cools to 25 °. Then ZnMB
S is dispersed in chilled oil and ground as in Example 2 until the degree of agriculture in oil is 10%. 15 somatotropins in the dispersion
Further mill until it has a median particle size no greater than micron. Each composition has an injection capacity of greater than 0.1 ml / sec.

Example 15 In this example a composition of the invention containing 42% bovine somatotropin is prepared by continuing to add somatotropin to the composition of Example 14 until each composition contains 42% somatotropin. Somatotropin while maintaining oil as a continuous phase
Disperse and mill as in Example 2 until it has a median particle size not greater than 15 microns. Each composition has an injection capacity of greater than 0.03 ml / sec.

Example 16 This example shows one of the following anti-hydrating agents: aluminum distearate or aluminum tristearate; mono-, di- or aluminum tripalmitylate or mono-, di- or aluminum trilaurate; mono- or magnesium distearate. , Mono- or di-magnesium laurate, or mono- or magnesium dipalmitylate; in Example 9 except that the mono- or calcium distearate, mono- or calcium dilaurate or mono- or dipalmitylate is replaced with AlMS. 1 illustrates a composition of the present invention containing 20% bovine somatotropin in the same oil used. The anti-hydrating agent is added to the oil prior to the addition of somatotropin. ZnMBS is added as in Example 2 until its concentration is 20%. Somatotropin 15 for dispersion and grinding
Continue until you have a median particle size no greater than micron. Each composition has an injection capacity of greater than 0.03 ml / sec.

Example 17 This example illustrates a composition of the present invention containing different concentrations of bovine somatotropin in the same oil as in Example 20. Example 20 uses an oil of the type used in Example 16 except that the somatotropin addition is continued until its concentration is 25%, 30% or 35%.
Dispersion and milling continue until somatotropin has a median particle size no greater than 15 microns. 0.03 for each composition
It has an injection capacity of more than ml seconds.

Example 18 When the bovine somatotropin used is chemically unbound or chemically bound to sodium or potassium cations, substantially the same results as in Examples 8-17 are obtained.

Example 19 Other bovine somatotropin, for example following amino acid _{sequence: NH 2 -met-phe (1} ) -pro (2) ... val (126) ... phe (1
_{90) -COOH NH 2 -ala-phe} (1) -pro (2) ... val (126) ... phe (1
_{90) -COOH NH 2 -ala-phe} (1) -pro (2) ... leu (126) ... phe (1
_{90) -COOH NH 2 -phe (1} ) -pro (2) ... leu (126) ... phe (190)
_{-COOH NH 2 -phe (1) -pro} (2) ... val (126) ... phe (190)
_{-COOH NH 2 -met-asp-glu} -phe (1) -pro (2) ... leu (12
6) ... phe (190) -COOH NH 2 -met-asp-glu-phe (1) -pro (2) ... val (12
6) ... phe (190) -COOH NH 2 -met (4) -leu (126) ... phe (190) -COOH NH 2 -met (4) -val (126) ... phe (190) having a -COOH Is used, substantially the same results as in Examples 8-18 are obtained.

Examples 20-30 When porcine somatotropin is used in place of bovine somatotropin in the treatments of Examples 8-18, the similarities of bovine and porcine somatotropin give substantially the same results.

While particular embodiments of this invention have been described, it will be apparent to those skilled in the art that various modifications can be made without departing from the true spirit and scope of this invention. Accordingly, the claims are intended to cover all modifications within the full inventive concept.

Claims (38)

[Claims] 1. A substantially non-aqueous injectable composition comprising a biologically active somatotropin and a biocompatible oil as the continuous phase of the composition, the composition being parenterally. To achieve a sustained release of biologically active somatotropin into the circulatory system of an animal, which is for administration and is characterized in that the amount of somatotropin is at least 10% by weight of the composition, Non-aqueous injectable composition. 2. The composition of claim 1 wherein said somatotropin is bovine or porcine somatotropin. 3. A composition according to claim 1 or 2 wherein said composition comprises 15% to 42% somatotropin. 4. The method according to claim 1, wherein somatotropin forms a complex with a non-toxic metal ion.
The composition according to any one of items 1 to 3. 5. The metal is zinc, sodium, potassium,
Iron, calcium, bismuth, barium, magnesium,
A composition according to claim 4 selected from manganese, aluminum, copper, cobalt, nickel and cadmium. 6. A composition according to claim 5, wherein the somatotropin is bovine somatotropin and the metal is zinc. 7. A composition according to claim 6 wherein the somatotropin is porcine somatotropin and the metal is copper. 8. A composition according to claim 1, wherein the somatotropin has a median particle volume diameter not greater than 15 micrometers. 9. The oil is corn oil, peanut oil, sesame oil,
Olive oil, palm oil, safflower oil, soybean oil,
9. Any one of claims 1 to 8 selected from cottonseed oil, rapeseed oil, and mixtures thereof.
The composition according to the item. 10. Somatotropin is in the form of a complex of somatotropin with a metal which is a reaction product of a metal ion selected from the group consisting of zinc and copper, the metal ion being 2 weight parts of this reaction product. A composition according to claim 1 comprising up to%. 11. The composition of claim 10 wherein the molar ratio of metal ion to somatotropin in the reaction product is 1: 1 to 4: 1. 12. The composition of claim 10 wherein said somatotropin is bovine or porcine somatotropin. 13. The method according to claim 1, wherein the somatotropin is bovine somatotropin and the metal ion is zinc.
The composition of paragraph 10. 14. The composition of claim 10 wherein the somatotropin is porcine somatotropin and the metal is copper. 15. A substantially non-aqueous composition for parenteral administration to animals comprising a biologically active somatotropin, an anti-hydrating agent, and a biocompatible oil as the continuous phase of the composition. Wherein the amount of somatotropin is at least 10% of the composition.
% Of the anti-hydration agent is at least 1% by weight of the composition to achieve a sustained release of biologically active somatotropin into the circulatory system of the animal. A non-aqueous injectable composition. 16. The composition of claim 15 wherein the anti-hydration agent is an aluminum, calcium or magnesium salt of a fatty acid containing 8 to 22 carbons. 17. The composition according to claim 16, wherein the fatty acid is a fatty acid selected from stearic acid, palmitic acid, lauric acid and mixtures thereof. 18. A weight ratio of somatotropin to anti-hydration agent of at least 1 in claims 15-17.
Item 10. The composition according to any one of items. 19. The method according to claim 15, wherein somatotropin forms a complex with a non-toxic metal ion.
Item 19. The composition according to any one of items 18 to 18. 20. The metal is zinc, sodium, potassium,
Iron, calcium, bismuth, barium, magnesium,
20. The composition of claim 19 selected from manganese, aluminum, copper, cobalt, nickel and cadmium. 21. The composition of claim 20 wherein the somatotropin is bovine somatotropin and the metal is zinc. 22. The composition of claim 21 wherein the somatotropin is porcine somatotropin and the metal is copper. 23. The composition of any one of claims 15 to 22 wherein somatotropin has an intermediate particle volume diameter not greater than 15 micrometers. 24. From claim 15 wherein the oil is selected from corn oil, peanut oil, sesame oil, olive oil, palm oil, safflower oil, soybean oil, cottonseed oil, rapeseed oil, and mixtures thereof. Item 23. The composition according to any one of items 23. 25. 15% to 42% by weight of bovine somatotropin, 2% to 5% by weight of aluminum mono- or distearate, and biocompatible vegetable oil as the continuous phase of the composition, the somatotropin comprising Has an intermediate particle volume diameter not greater than 10 micrometers,
The weight ratio to stearate is at least 3,
16. The substantially non-aqueous injectable composition of claim 15 for parenteral administration to humans. 26. The composition of claim 25, wherein somatotropin forms a complex with zinc. 27. Somatotropin is in the form of a complex of a metal and somatotropin, which is a reaction product of a metal ion selected from the group consisting of zinc and copper, and the metal ion is contained in the reaction product. The composition according to claim 15, wherein the composition comprises up to% by weight. 28. The composition of claim 27, wherein the molar ratio of metal ion to somatotropin in the reaction product is 1: 1 to 4: 1. 29. The composition of claim 27, wherein said somatotropin is bovine or porcine somatotropin. 30. The somatotropin is bovine somatotropin and the metal ion is zinc.
The composition of paragraph 27. 31. The composition of claim 27, wherein the somatotropin is porcine somatotropin and the metal is copper. 32. A substantially non-aqueous injection comprising a biologically active somatotropin, a water soluble compound of a metal ion selected from zinc and copper, and a biocompatible oil as the continuous phase of the composition. A possible composition, wherein the composition is for parenteral administration and the amount of somatotropin is at least 10% by weight of the composition and the metal ions are up to 2% by weight of the composition. A substantially non-aqueous injectable composition for achieving sustained release of a highly active somatotropin into the circulatory system of an animal. 33. The composition of claim 32, wherein the molar ratio of metal ion to somatotropin is about 1: 1 to 4: 1. 34. The composition of claim 32, wherein said somatotropin is bovine or porcine somatotropin. 35. The method according to claim 32, wherein the somatotropin is bovine somatotropin and the metal ion is zinc.
The composition of paragraph. 36. The composition of claim 32, wherein the somatotropin is porcine somatotropin and the metal ion is copper. 37. A method for enhancing lean-fat ratio, dietary efficiency or milk production of an animal comprising the step of biologically active somatotropin and a biocompatible oil as the continuous phase of the composition. A non-aqueous injectable composition of an animal of biologically active somatotropin, wherein the composition is for parenteral administration and the amount of somatotropin is at least 10% by weight of the composition. The above method, characterized in that the animal is parenterally administered with a substantially non-aqueous injectable composition to achieve sustained release into the circulatory system. 38. The method of claim 37, wherein the animal is selected from cattle, sheep, goats, and pigs.